Anal. Chem. 1995, 67, 4184-4189
Microchip Separations of Neutral Species via Micellar Electrokinetic Capillary Chromatography Alvin W. Moore, Jr., Stephen C. Jacobson, and J. Michael Ramsey* Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Building 45OOS, MS 6142, Oak Ridge, Tennessee 3783 1-6142
Micellar electrokineticcapillary chromatography (MECC) of three neutral coumarin dyes was perfonned on glass microchips. Manifolds of channels for analyte injection and separation were machined into one surface of the glass substrates using standard photolithographic, etching, and deposition techniques. Coverplates were then directly bonded Over these channels to form capillary networks, with fluid flow in these networks controlled by varying the applied high-voltage potentials at the outlets. 'Ihe separation capillarywas 16.5 cm long for a serpentine channel chip and 1.3 cm long for a straight channel chip. Detection of analyte zones was accomplished by laserinduced fluorescence using the UV lines (-350 nm) of an argon ion laser. At low applied electric field strengths, MECC analyses with on-chip injections gave high reproducibilities in peak areas and migration times (< 1%for two of the three coumarins) and near constant separation eillcienciesthroughout the analysis. At high fields (>400 V/cm), analysis times were shorter, but separation efficiency decreased at later migration times. 'Ihese peaks showed significant broadening, consistent with mass transfer effects. Many of today's applications of analytical chemistry involve collection of samples from some remote site, transportation of these samples back to the laboratory, and analysis using benchtop instrumentation. These situations range from detection of pollutants in water supplies to process monitoring for quality control. In the typical analytical laboratory, a universal detection system such as a UV absorption detector and a separation technique such as liquid chromatography (LC) or capillary electrophoresis (CE) are used to quantify the analyte of interest. The separation method provides the selectivity between components and classes of components that the universal detector lacks. If the analysts' needs change, such that they must quantify other species, the selectivity can be easily modified by changing the separation conditions such as the pH of the mobile phase or buffer, the organic solvent content, or the elution gradient. The enhanced versatility of this method comes at the expense of requiring that samples be collected and transported to the lab. An attractive means of saving time and expense would be to take the instrument to the sample. Micromachining technology may allow the ultimate miniaturization of chemical measurement instrumentation. Liquid phase separation devices are particularly amenable to miniaturization because analytical separation performance often improves with decreasing size of the components involved. CE is a relatively new separation method which has 4184
Analytical Chemistry, Vol. 67, No. 22, November 75, 7995
used microcolumn capillaries from its inception. Because CE gives highefficiency separations but requires no high-pressure pump or gas supply, it is especially well suited for miniaturization. Substantial progress has already been made in this A problem with CE for general analysis is its inability to separate uncharged species. All neutral species in a particular sample will have zero electrophoretic mobility and thus the same migration time. Previously, our group has investigated openchannel electrochromatography (OCEC) on a microchip as a means of separating neutrals.5 In this previous work, sample components were separated by their partitioning interaction with a stationq phase coated on the channel walls. The mobile phase was driven not by a conventional pump but by electroosmoticflow. Micellar electrokinetic capillary chromatography (MECC) is a operational mode of CE which was developed by Terabe et aL6to address the separation of neutrals by CE. A surfactant such as sodium dodecyl sulfate (SDS) is added to the CE buffer in sufficient concentration to form micelles in the buffer. In a typical experimental arrangement, the micelles move much more slowly toward the cathode than does the surrounding buffer solution. The partitioning of solutes between the micelles and the surrounding buffer solution provides a separation mechanism similar to that of LC. In this work, MECC was performed in a capillary etched into the surface of a glass chip. All of the fluidic manipulation necessary for sample injection and analysis was done through a manifold of channels, with flow controlled by the voltage applied to the reservoir at the end of each channel. Laser-induced fluorescence detection was used to monitor the separations. Some results of this implementation of MECC will be shown, and some of its advantages and disadvantages for chigbased microinstrumentation will be discussed. OVERVIEW An in-depth treatment of the theory of MECC has been given and is beyond the scope of this paper. Only a brief by (1) Harrison, D. J.; Manz, A; Fan, Z.; Liidi, H.; Widmer, H. M. Anal. Chem. 1992,64,1926-1932. (2) Seiler, K; Harrison, D. J.; Mmz, A Anal. Chem. 1993,65,1481-1488. (3) Jacobson, S. C.; Hergenroder, R; Koutny, L. B.; Warmack, R J.; Ramsey, J. M. Anal. Chem. 1994,66,1107-1113. (4) Jacobson, S . C.; Hergenroder, R; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994,66,1114-1118. (5) Jacobson, S . C.; Hergenroder, R; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994,66,2369-2373. (6) Terabe. S.; Otsuka, IC; Ando, T. Anal. Chem. 1985,57,834-841. (7) Sepaniak, M. J.; Powell, A C.; Swaile, D. F.; Cole, R 0. In Capillmy Electrophoresis: l%eo?y and Practice; Grossman, P. D., Colbum, J. C., Eds.; Academic Press, Inc.: San Diego, CA, 1992 pp 159-89. (8) Sepaniak, M. J.; Cole, R. 0. Anal. Chem. 1987,59,472-476.
0003-2700/95/0367-4184$9.00/0 0 1995 American Chemical Society
review will be given here. For neutral solutes, the separation mechanism is essentially chromatographic and can therefore be described with modified chromatographic relationships. The capacity factor, k', for a given solute and experimental conditions is the ratio of the total moles of solute in the stationary phase (here, the pseudostationaryphase of the micelles) to those in the mobile phase. For MECC, k' is modified to account for the movement of the micelles,
where t~is the solute retention time, to is the "void time" (retention time for a solute moving at the rate of the electroosmotic flow), tm is the micelle retention time (i.e., retention time of a completely retained solute), K is the partition coefficient, V, is the volume of the micellar phase, and V, is the volume of the mobile phase. If the micelles were indeed stationary, t,,, would become infinite and the equation would reduce to its conventional form for chromatography. Because to is the retention time for a solute moving with the electroosmoticflow, and tmis that of a solute completely retained in the micelles, neutral solutes must elute between to and t,. Resolution can be improved by increasing the difference between to and tm,thus increasing the "window" of time over which sample components can elute. Resolution can also be improved by changing the k' values of the solutes, which affects both the retention and the selectivity of the separation. This can be done in many of the same ways it is done in liquid chromatography, such as by adjusting the temperature, buffer concentration, or pH, or by the addition of organic modifiers. Organic solvents such as methanol and acetonitrile can have multiple effects in that they may modify the electroosmotic flow in the system or alter the hydrophobic interactions between solute and micelle, but they also affect micelle structural interactions and so alter the partitioning kinetics. Changes in the organic solvent content of the buffer also modify the electroosmotic flow in the system. EXPERIMENTAL SECTION
The microchips were constructed using standard photolithographic, wet chemical etching and bonding techniques described previously? An ordinary soda lime glass microscope slide was used as a substrate, into which was etched a network of open channels (see Figure 1). The serpentine channel geometry (Figure la) allowed use of a longer separation capillary within a small area, while the straight channel geometry (Figure lb) allowed experiments with high applied electric field strengths. These channel networks were closed with thin coverplates, directly bonded to the substrate, to form capillary networks. Cylindrical glass reservoirs were then bonded with epoxy to the capillary outlets. The lengths of the capillary channels may vary with the placement of the coverplate. Figure 1gives the channel lengths for the two chips used in this work. Use of the wet chemical etch on a glass substrate results in an isotropic etch. That is, the glass etches at the same rate in all directions, and the resulting channel geometry is trapezoidal? The channel cross section dimensions were the same for both chips used in this (9) Jacobson, S. C . ; Koutny, L. B.; Hergenroder, R; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476.
l o mm
glau, subtrate
€
10 mm
t
glass substrate
Figure 1. Schematic diagrams of microchip geometry. The large outer boxes show the outlines of the glass substrates. Individual capillary lengths are measured from the point of injection to the point where the capillary passes beyond the cover plate into the respective reservoir. (a) The serpentine channel geometry used for the majority of the work, with channel lengths as follows: analyte channel, 9.2 mm; buffer channel, 6.6 mm; analyte waste channel, 7.7 mm; waste (separation) channel, 171 mm. (b) The straight channel geometry used for the high-field experiments, with channel lengths as follows: analyte channel, 8.0 mm; buffer channel, 4.6 mm; analyte waste channel, 9.8 mm; waste (separation) channel, 17.2 mm.
work. The channels were 10pm deep, 60 pm wide at the bottom, and 80 pm wide at the top. These dimensions were measured with a profilometer (Alpha-Step 200, Tencor Instruments, Mountain view, CA) after bonding the coverplate but before attaching the reservoirs. Separation of the analyte zones was monitored via laser-induced fluorescence using an argon ion laser (351.1-363.8 nm, all lines; -50 mW Coherent Innova 90,5 W Palo Alto, CA) for excitation. The UV beam from the laser was prefiltered with a Coming 7-37 colored glass filter (blue, narrow bandpass; transmittance, 30%at 360 nm) to reduce plasma emissions and then focused at the desired point on the capillary with a planoconvex fused silica lens (focal length, 100 mm; Newport Corp., Irvine, CA). The laser impinged on the chip at an angle of 45" with the surface. The fluorescence signal was collected from below the chip by a 21 x microscope objective (Bausch & Lomb Opt. Co., Rochester, NY), filtered with a Coming 2-73 colored glass filter (yellow, sharp cuton at 426 nm), and detected with a photomultiplier tube (PMT Oriel 77340, Stratford, 0. The PMT current was amplified and converted to a proportional voltage with a Keithley 617 electrometer (Keithley Instruments Inc., Cleveland, OH). The analog voltage output of the electrometer was measured with a multifunction interface board (AT-MIO-16X, National Instruments, Austin, TX) controlled by software written in-house using LabVIEW 3.0 for Windows (National Instruments, Austin, TX) on a PC compatible computer. Separation efficiencies were obtained by calculation of peak statistical moments, also in LabVIEW. Platinum electrodes in each reservoir provided electrical contact between the buffer solutions and the CE high-voltage power supply (CZElOOOR, Spellman Inc., Plainview, NY). A Analytical Chemistty, Vol. 67, No. 22, November 15, 1995
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voltage dividerhelay apparatus described earlier1° was used to set the relative voltages applied to each reservoir and to switch between run and inject modes under computer control. For both chip geometries, the separation voltage was applied over the entire length of the capillary channel for all analyses. However, for individual groups of analyses, the length of the separation channel used was set by the location of the point of detection. The reservoirs at the end of each capillary channel had a volume of only 200 pL. To avoid problems with changes in buffer concentration due to evaporation, the reservoirs were sealed with thin rubber septa held in place with paralilm. The platinum electrodes could be inserted through the septa if the septa was initially pierced with a syringe needle. The reservoirs could be flushed and filled a in similar manner with a syringe of buffer or sample. In use, the seal formed around the electrodes was almost gas-tight. The resolution between peaks could be maintained from run to run for several hours before significant evaporation effects were noticed. Only the data in Figure 3, as mentioned below, were acquired with open buffer reservoirs. The analytes used in these experiments were the neutral dyes coumarin 440 (C440), coumarin 450 (C450), and coumarin 460 (C460, Exciton Inc.). Individual stock solutions of each dye were prepared in methanol and then diluted in the analysis buffer before use. The concentration of each dye was -50 pM unless indicated otherwise. The substrate is glass rather than quartz, so there is substantial background fluorescence where the UV laser strikes the chip. No attempt was made to measure limits of detection with the present system. The MECC buffer was composed of 10 mM sodium borate @Hgal),50 mh4 SDS, and 10%(v/v) methanol. The methanol aids in solubilizing the coumarin dyes in the aqueous buffer system and also affects the partitioning of some of the dyes into the micelles. Due care must be used in working with coumarin dyes as the chemical, physical, and toxicological properties of these dyes have not been fully investigated.” Sample Injection. The microchips were operated in a “pinched injection” mode described previ~usly.~The voltages applied to the reservoirs are set to either an “inject” (sample loading) or a “run” (separation) configuration. In the inject mode, a frontal chromatogram of the solution in the analyte reservoir is pumped electroosmotically through the intersection and into the analyte waste reservoir. Voltages applied to the buffer and waste reservoirs also cause weak flows into the intersection from the sides and then into the analyte waste reservoir. These flows serve to confne the stream from the analyte reservoir to give a welldefined plug of sample in the intersection. The chip remains in this mode until the slowest moving component of the sample has passed through the intersection. At this point, the sample plug in the intersection is representative of the analyte solution, with no electrokinetic bias. An injection is made by switching the chip to the run mode, which changes the voltages applied to the reservoirs such that buffer now flows from the buffer reservoir through the intersection, into the separation column, and eventually into the waste reservoir. The plug of sample which was previously in the intersection is swept onto the separation column. Proportionately lower voltages are applied to the analyte and analyte waste reservoirs to cause a weak flow of buffer from the buffer reservoir (10) Jacobson, S. C.; Hergenroder, R; Moore, A W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66,4127-4132.
(11) Exciton, Inc., Dayton, OH, 1991.
4186 Analytical Chemistry, Vol. 67, No. 22, November 75, 7995
3
4
5
6
7
8
Time (min)
Figure 2. Microchip MECC analysis of a mixture of coumarin dyes. The small peak at 4.6 min is an unidentified impurity in one of the dyes. Concentration of each dye was -50 pM. Other analysis conditions are given in the text.
into these channels. These flows ensure that the sample plug is cleanly “broken off from the analyte stream and that no excess analyte leaks into the separation channel during the analysis. This sample loading method is time-independent (after the initial time necessary to pump all components through the intersection), nonbiased, and reproducible. RESULTS AND DISCUSSION The results of an MECC analysis of a mixture of C440, C450, and C460 are shown in Figure 2. The peaks were identified by individual analyses of each dye. The migration time stability of the first peak, C440, with changing methanol concentration (see below) was a strong indicator that this dye did not partition into the micelles to a significant extent. Therefore, it was considered an electroosmotic flow marker with migration time to. The last peak, C460, was assumed to be a marker for the micellar migration time, tm. Using these values of to and tm from the data in Figure 2, the calculated elution range, to/tm, is 0.43. This agrees well with a literature value of t0/tm = 0.4 for a similar buffer system* and supports our assumption. This analysis was done in the serpentine chip with a separation length of 21.3 mm and an applied electric field strength of 47 V/cm. Buffer Evaporation. Figure 3 shows several successive analyses of the same three component sample over a period of 30 min. These analyses were done with an applied field of 96 V/cm and a separation length of 17 mm. Notice that from parts a to c of Figure 3, the C440 peak and the C450/C460 peak pair tend to move closer together (the elution range decreases), and the resolution between the C450 and C460 peaks gradually decreases. The high voltage was switched off for 5 min between parts b and c of Figure 3, so that if gradual heating of the MECC buffer in the chip were responsible for the lost resolution, it should have been partially restored in Figure 3c. Obviously, this was not the case. Immediately before the analysis shown in Figure 3d, the buffer and sample solutions were replaced. Notice that the resolution between the last two peaks has been restored, indicating some change in the buffer solutions during the course of the analyses shown in Figure 3a-c. For the data in Figure 3, the reservoirs at the end of each capillary channel were open to the surrounding air. When the
Table 1. Peak Parameters.
migration time (s)
f8
G
i 1.5
I
2.0
3.0 lime (min)
2.5
3.5
4.0
4.5
-
B1.5
2.0
3.5
3.0
2.5
4.0 b,
15 min 4.5
:
Time (min)
av (n = 5) SD %RSD
209.1 1.94 0.93
av (n = 5) SD %RSD
384.6 3.27 0.85
av (n = 5)
438.1 5.76 1.31
SD %RSD
peak
area
theoretical plates (N)
HETP (um)
3523 60 1.71
6.05 0.10 1.71
3855 80 2.09
5.53 0.12 2.09
C440 2.82 0.03 0.91 C450 4.92 0.05 0.92 c460 7.62 0.17 2.21
3705 101 2.73
~
5.75 0.16 2.78
a Data for five replicate analyses w ith applied electric field of 47 V/cm, separation length of 21.3 mm.
Table 2. Ratio Peak Parameters.
migration ratio Ci40/ time C460/ 1.5
2:o
3:s
2.5
4.0
4.5
Time (min)
q\ ,
av (n = 5) SD d, 35 min (after replacing buffer)
A
f
E.
d
1.5
,,... ,
2.0
2.5
, ,
.
J,
, 3.0 Time (min)
%RSD
peak areaC460/ ratio C440/
factor capably (k3, nn..nn:kr
C450
C450
C450
C450
C450
0.544 0.01 1.57
1.14 0.01 0.54
0.574 0.01 1.11
1.55 0.02 1.51
6.89 0.05 0.78
0 Ratio values and capacity factor for C450 calculated from the data used in Table 1.
3.5
4.0
.-4.5
Figure 3. Effects of methanol evaporation in microchip MECC. The
times indicated in the figure are times between the ends of the
analyses, immediately after acquisition when the data were saved. Peaks are identified as in Figure 2. buffers were entirely aqueous, buffer evaporation was not a significant problem. However, the MECC buffer contained 10% (v/v) methanol, so methanol evaporation might be significant. A decrease in the methanol concentration would cause the C450 to prefer the free buffer solution less and partition to a greater extent into the micelles. That is, the partition coefficient of the C450 would increase, and the selectivity between C450 and C460 would decrease, lowering the resolution between them. Also, addition of methanol is known to extend the elution range in MECC? so the decrease seen in Figure 3a-c agrees well with a decrease in methanol concentration due to evaporation. Weinberger and Lurie12 have suggested that similar evaporation problems with MECC buffers containing organic modifiers contribute to poor run-to” reproduciblity. Reproducibility. The results for five replicate analyses of the three coumarin dyes mix as in Figure 2 are shown in Table 1. The migration times, peak areas, and peak variances were obtained from the best-fit values for Gaussian curves fitted to the experimental data. The percent relative standard deviation (%RSD) of the migration times and peak areas are slightly lower than expected for MECC. Some authors have reported %RSD of migration times of 2%or l e s ~ , with ~ J ~some as high as 10%over the course of a day,13 while %RSD values for peak area are (12) Weinberger, R;Lune, I. Anal. Chem. 1991,63,823-827. (13) Northrop, D.M.; Matire, D.E.;MacCrehan, W. A Anal. Chem. 1991,63, 1038-1042.
generally even higher. Here, for the C440 and C450 peaks, peak area %RSD values are
